Improving sequence-specific antimicrobials by blocking dna repair

ABSTRACT

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

FIELD OF THE INVENTION

The invention relates to endonuclease-based antimicrobials that generatedouble-strand break(s) (DSB(s)) in prokaryotic cells. In this respect,the invention especially concerns a method involving blocking DNA repairafter a nucleic acid has been submitted to DSB. The invention alsorelates to a vector encoding such endonuclease and a protein blockingDNA repair, a pharmaceutical composition and a product comprising saidvector for use in the treatment of diseases dues to a bacteriuminfection

BACKGROUND OF THE INVENTION

Cas proteins such as Cas9, of CRISPR-Cas systems, are members of theprogrammable nucleases, that have emerged as popular tools to introducemutations in eukaryotic genomes as also are Zinc Finger Nucleases (ZFN)or Transcription Activator-Like Effector Nucleases (TALEN). Doublestrand breaks introduced in genomes by these nucleases can be repairedeither through Homology Directed Repair (HDR) or through Non-HomologousEnd Joining (NHEJ). Most bacterial species lack a Non-Homologous EndJoining (NHEJ) system. When a double strand beak is introduced at agiven position in all copies of the chromosome simultaneously, thebacterium will die without DNA repair. When a double strand beak isintroduced at a given position in all copies of an antibiotic resistanceplasmid simultaneously, the bacterium will be susceptible to theantibiotic without DNA repair.

In bacteria, double strand breaks are generally repaired throughhomologous recombination with an intact sister chromosome. The firststep of repair involves loading of the RecBCD or AddAB complex on thedouble strand ends. The ends are then resected through a helicase andexonuclease activity until a specific sequence motif known as the chisite is found. In E. coli the sequence of the chi site is GCTGGTGG. Oncea chi site is found, the RecBCD/AddAB complex keeps degrading one of thestrands while the other strand is loaded with the recA protein. Thenucleoprotein filament can then invade the sister chromosome andinitiate replication dependent repair. RecBCD/AddAB resects doublestranded ends present in the cell at the very high speed of ˜1 kb/sec.If no homologous sequence is present in the cell the DNA molecule iscompletely destroyed. Upon infection, phages thus need to protect theirdouble strand ends from RecBCD/AddAB. For these purpose they haveevolved different strategies to either block the access of the doublestrand end (e.g. the Mu Gam protein) d'Adda di Fagagna et al., EMBOreports, 4(1):47-52 (2003), or directly block the activity ofRecBCD/AddAB through direct binding (e.g. the lambda Gam protein).Murphy et al., J. Bacteriology 173 (18): 5808-5821 (1991).

It was shown in the prior art that nuclease cleavage can kill the cellswhen all chromosomal copies are cut simultaneously and no intacttemplate is available for homology directed repair. However, not alltargets are equal and some positions are being targeted more efficientlythan others. Inefficient nuclease interference can be tolerated throughcontinuous repair by the homologous recombination pathway. Accordingly,in several bacteria a DNA repair occurs after nuclease cleavage.Thereof, the use of the nucleases only is not sufficient to killbacteria.

Consequently, there is a need to novel method allowing efficientlykilling of bacteria and thus being used in antimicrobial treatments.

SUMMARY OF THE INVENTION

Surprisingly, the inventors of the present invention found thatcombining the action of an endonuclease with the action of some proteinsinvolved in bacteriophage DNA protection enhance the ability ofendonuclease to kill bacteria cells since these proteins do not allowDNA repair.

According to a first aspect, the invention thus relates to a method forkilling a bacterium comprising contacting the bacterium with anendonuclease, preferably encoded by at least one recombinant phagemid(s)or plasmid(s), that creates a double-stranded break in the chromosomalDNA of the bacterium and an exogenous molecule that inhibitsdouble-stranded break repair, preferably a protein that binds to theends of the double-stranded break.

Using the method of the present application, it is possible to selectspecific DNA sites for the cleavage. Such site may be the part of theDNA sequences responsible for the antibiotic resistance of bacterium.

According to another aspect, the method of the invention is used formaking a bacterium more susceptible to an antibiotic, said methodcomprising contacting the bacterium with an endonuclease, preferablyencoded by at least recombinant phagemid(s) or plasmid(s), and theantibiotic, wherein the endonuclease creates a double-stranded break inan antibiotic resistance gene encoded by the bacterium, and an exogenousmolecule that inhibits double-stranded break repair, preferably aprotein that binds to the ends of the double-stranded break. In oneembodiment, the recombinant phagemid or plasmid encodes a Cas9 nuclease,a guide RNA, and an exogenous Gam protein.

In order to implement the method of the invention, it is necessary toprovide a vector, particularly a phagemid vector encoding a nucleasesusceptible to cleave DNA double strand of bacterium and a protein thatbinds to the ends of the double-stranded break and inhibit DSB repair.

According to one aspect, the invention thus relates to a phagemid vectorencoding a nuclease, and optionally, an exogenous protein that binds tothe ends of the double-stranded break and inhibit DSB repair.

In various embodiments, the invention relates to a phagemid vectorencoding a nuclease, preferably Cas9 nuclease, a guide RNA, and anexogenous protein that binds to the ends of the double-stranded breakand inhibit DSB repair, particularly Gam protein. In another embodiment,the guide RNA targets an antibiotic resistance plasmid or a plasmidcarrying virulence genes. In various embodiments, the guide RNA targetsthe bacterial chromosome. In various embodiments, the phagemid vector isa P1 bacteriophage. In various embodiments, the phagemid vector is a λbacteriophage.

According to another aspect, the invention also relates to a host cellcomprising the phagemid or plasmid vector of the invention and aphagemid or plasmid vector encoding the protein inhibiting DSB repair.

According to a further aspect, the invention also relates to apharmaceutical composition comprising the phagemid or plasmid vector ofthe invention and a vector encoding the protein inhibiting DSB repair orthe protein inhibiting DSB repair and a pharmaceutical acceptablevehicle for use in the treatment of diseases due to a bacteriuminfection.

The present application also relates to a product comprising

-   -   at least one phagemid or plasmid vector or pharmaceutical        composition of the invention, and    -   at least another therapeutic agent, in particular an antibiotic

as a combination product for simultaneous, separate or sequential usefor the treatment of at least one disease due to a bacterium infection,particularly infection due to at least one of bacteria selected from thegroup comprising of Enterobacter, Streptococci, Staphylococci,Enterococci, Salmonella, Pseudomonas, Mycobacterium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D. Weak self-targeting CRISPR-Cas9 systems can be toleratedthrough homology directed repair. (A) Position of the targets on the E.coli chromosome. Targets on the inside of the circle are on thenon-template strand, targets on the outside are on the template strand.(B) The pCRRNA carrying different spacers was transformed in cellsexpressing Cas9 constitutively. Average CFU numbers are reported fortransformation in wild-type cells (black bars) and recA− cells (whitebars), showing that some spacers can be tolerated in the presence ofrecA but not in the recA− strain (mean±s.d., n≥3). Transformation eventsyielding small colonies are marked with a star. (C) Schematics of thetransformation assay performed to demonstrate homology directed repair.The pCas9 (also designated pCas9-a carrying a control spacer that can beeasily replaced through restriction-ligation cloning) plasmid SEQ ID NO:60 (indicated as SEQ ID No. 117 in the priority application) carryingCas9, the tracrRNA and a CRISPR array was programmed to target aposition within the lacZ gene. The resulting plasmid pCas9::lacZ2(carrying a spacer targeting the lacZ gene) having the sequence of SEQID No. 119 was transformed in cells carrying a plasmid with homologiesto the target region but carrying a mutation preventing Cas9 cleavage(pLCX SEQ ID NO: 66). (D) CFU numbers are reported after transformationeither in wild-type (black bars) or recA− cells (white bars), showingthat the presence of a repair template rescues killing induced by Cas9cleavage of the lacZ2 target (mean±s.d., n≥13).

FIG. 2: Colony size after transformation with self-targeting CRISPRsystems. The pCRRNA plasmid carrying different spacers was transformedin MG1655 cells expressing Cas9 constitutively from plasmid pCas9. Cellswere plated on selective medium and colony diameter was quantified after16H of incubation at 37° C. using the ImageJ software. A minimum of 50colonies were counted for each individual transformation.

FIG. 3A-C: Cas9 cleavage in the chromosome induces the SOS response. (A)The pCRRNA plasmid programmed to target the lacZ1 position (black bars)or a control empty pCRRNA (white bars) were introduced in cellsexpressing Cas9 under the leaky control of a non-induced ptet promoterin the chromosome. SOS induction is reported by a GFPmut2 gene under thecontrol of the sulA promoter (pZA31-sulA-GFP). GFP fluorescence wasmeasured during exponential growth (mean±s.d., n≥3). (B) SOS responseinduced by targeting with different spacers. The bar marked as “control”indicates the auto-fluorescence level of E. coli without thepZA31-sulA-GFP plasmid. Spacers that cannot be transformed underconstitutive Cas9 expression from the pCas9 (see FIG. 1B) are shown inwhite. Spacers that can be transformed but lead to the formation ofsmall colonies (see FIG. 1B) are shown in grey. Finally, spacers thatcan be transformed in the presence of pCas9 and form colonies of regularsize (see FIG. 1B) are shown in black (mean±s.d., n≥3). (C) analysis ofCas 9 induced deletions in recB-strain: the deletions observed aftertransformation of the stain are indicated.

FIG. 4: SOS activation by Cas9 cleavage of the lacZ1 target with orwithout anhydrotetracyclin (aTc) induction. The pZA31-sulA-GFP plasmidwas used to monitor SOS induction after pCRRNA::Ø or pCRRNA::lacZ1transformation in LCE03 cells expressing cas9 under the control of aptet promoter in the chromosome (see Table 1). Cells were grown to an ODof 0.4 and 1 uM aTc was added. GFP fluorescence was measured 2H afterinduction. The strong GFP signal measured in the absence of aTcindicates that the ptet promoter controlling Cas9 is leaky.

FIG. 5: Map of plasmid psgRNAc BsaI SEQ ID NO: 62 (indicated as SEQ IDNo. 123 in the priority application)

FIG. 6A-B: Gam can block DNA repair of double strand breaks introducedby Cas9. A) Representation of possible outcomes of Cas9 cleavage in thepresence or absence of Gam. Upon targeting by weak spacers or in anyother situation where a homologous template molecule is present in thecell, Cas9 breaks can be repaired through homology directed repair(HDR). In E. coli this can be achieved by the recBCD homologousrecombination pathway. In the presence of Gam, DNA ends are protectedfrom the action of recombinases. The presence of unrepaired DNA in thecell will ultimately lead to cell death. B) The pCas9 plasmid carryingeither an empty CRISPR array, the lacZ1 spacer or the lacZ2 spacer wastransformed in cells containing the pLC13 plasmid which carries the Mugam gene under the control of a pBAD promoter. Transformants were platedon selective medium either with or without arabinose (−ara/+ara). Thenumber of colony forming units is reported. Error bars represent thestandard deviation of three independent assays.

FIG. 7: pPhIF-Cas9 plasmid map (SEQ ID NO: 68)

FIG. 8: pBAD-MuGam plasmid map (SEQ ID NO: 69)

FIG. 9: MuGam RBS library (selection). Black squares mark selectedclones for further characterization. The RBS sequence upstream of themu-gam gene in pBAD-MuGam was modified by running an iPCR reaction onthe plasmid followed by a one-pot phosphorylation-ligation reaction. Thereligated plasmids were co-transformed into MG1655 cells containing thepPhIF-Cas9 plasmid, plated in LB-agar supplemented with 50 μg/mLkanamycin, 100 μg/mL chloramphenicol, 0.1 mM IPTG and 40 μg/mL X-gal andgrown for 20 hours at 30° C. Next, 95 single colonies were selected andgrown in 500 μL LB supplemented with 50 μg/mL kanamycin and 100 μg/mLchloramphenicol in 96-deep-well plates for 18 hours at 1000 rpm at 30°C. Next day, each culture was diluted 1:100 in distilled water. Thecells were assayed in four conditions: plates without inducer; platesthat contained 5 mM arabinose; plates that contained 0.1 mM DAPG; andplates that contained both 5 mM arabinose and 0.1 mM DAPG. Thisexperiment allows for the comparison of cell morphology and/or toxicityin the presence of Mu-Gam only and its effects when Cas9-sgRNA isco-expressed. Highlighted RBS library hits (black rectangles) showsdying colonies upon induction of Cas9 and Mu-Gam.

FIG. 10: CFUs of droplet dilutions of selected MuGam RBS clone. Oneclone were selected for its potential Mu_Gam adjuvant activity and amore detailed characterization was performed on LB-agar plates in theparticular conditions (no inducer; plus arabinose; plus DAPG; plus DAPGand arabinose). After an additional 24-hour incubation period CFUs werecounted. For the “+DAPG, +Ara” dataset, colonies were directly countedfrom the undiluted droplet. For the “+DAPG” dataset, colonies werecounted at 10⁻² and 10⁻³ dilutions, the dilution factor calculated andthe number of CFUs in the undiluted droplet estimated. For “No inducer”and “+Ara” conditions, the number of CFUs in the undiluted droplet wasestimated by counting the number of colonies in the 10⁻⁵ and 10⁻⁶dilutions and calculation the dilution factor.

FIG. 11: Activity of MuGam in a non-targeted sgRNA background. Cellscontaining pBAD-MuGam hit were co-transformed with a pPhIF-Cas9 variantwith a non-targeted sgRNA sequence. Cells were analyzed by the dropletmethod as explained in (A) and CFUs counted. To estimate the CFUs in theundiluted droplet, CFUs were counted at the 10⁻⁶ and 10⁻⁵ dilutions, thedilution factor calculated and the number of CFUs in the undiluteddroplet calculated. No toxicity of MuGam can be observed in the absenceof Cas9 targeting in the chromosome.

FIG. 12: pBAD-LambdaGam plasmid map (SEQ ID NO: 72).

FIG. 13: pCas9-MuGam/LambdaGam plasmid map (SEQ ID NO: 71/SEQ ID NO:72).

DETAILED DESCRIPTION OF THE INVENTION

In the aim to avoid bacterium DNA sequence repair after nucleasecleavage, the inventor found that specific proteins that bind the end ofcleaved site may be used. The inventors thus implemented a method forkilling bacterium comprising contacting the bacterium with anendonuclease, preferably encoded by a recombinant phagemid(s) orplasmid(s), wherein the recombinant phagemid(s) or plasmid(s) encodes anendonuclease that creates a double-stranded break in the chromosomal DNAof the bacterium, and an exogenous molecule that inhibits DNA repair.

In a preferred embodiment, the molecule is an exogenous protein thatbinds to the ends of the double-stranded break and inhibits DSB repair.

In another embodiment, the exogenous protein does not bind to the endsof the double strand break but affects other repairing mechanism,preferably recBCD.

In a particular embodiment, the method encompasses generating adouble-strand break (DSB) in the chromosomal DNA of the cell using achemical reagent such as nuclease, in particular a meganuclease selectedfrom a Homing endonuceases (HEs) or an artificial endonuclease selectedfrom the group comprising or consisting of a Zinc Finger Nuclease, TALENand a CRISPR-Cas system, or using a physical reagent such asirradiation, or expressing said chemical reagent in the cell as a resultof expression of a polynucleotide encoding the same when said cell hasbeen genetically transformed with said polynucleotide.

In one embodiment, the endonuclease specifically cleaves the chromosomalor extrachromosomal DNA of the bacterium at less than 2, 3, 4, 5, 6, 7,8, 9, or 10 different sites. Most preferably, the endonucleasespecifically cleaves the chromosomal or extrachromosomal DNA of thebacterium at a single site.

In another embodiment of the invention, the protein which binds cleavedends of DNA and block in such way DNA repair is selected from the groupcomprising or consisting of Mu phage Gam protein, a lambda phage Gamprotein, or a phage T7 gp5.9 protein. Preferably, the protein is arecBCD or AddAB inhibitor. Other inhibitors of recBCD or AddAB are knownin the art [43] In various embodiments, the bacterium comprises a recBCDhomologous repair pathway or an AddAB system. In various embodiments,the bacterium does not comprise a recBCD homologous repair pathway or anAddAB system.

In the present invention a programmable nucleases and in particular theCRISPR-Cas9 system can be used as a sequence specific antimicrobial whendelivered in bacterial populations [15] This system relies on theability of the RNA-guided Cas9 nuclease to kill bacteria whenintroducing a double strand break in the chromosome. However, somebacterial DNA repair pathways can compete with Cas9 cleavage allowingcells to survive. The recBCD homologous repair pathway can indeed repairbreaks introduced when Cas9 is guided by weak guide RNAs that do notlead to the simultaneous cleavage of all copies of the target sequence,leaving an intact copy of the target sequence available as a repairtemplate at any given time.

The term “CRISPR” or “Clustered regularly interspaced short palindromicrepeats” as used in the present invention relates to segments ofprokaryotic DNA containing short repetitions of base sequences. Eachrepetition is followed by short segments of “spacer DNA” from previousexposures to a bacteriophage virus or plasmid.

The term “CRISPR/Cas9 system” as used in the present invention relatesto a prokaryotic immune system that confers resistance to foreigngenetic elements such as those present within plasmids and phages andprovides a form of acquired immunity. CRISPR spacers recognize and cutthese exogenous genetic elements in a manner analogous to RNAinterference in eukaryotic organisms. By delivering the Cas9 nucleaseand appropriate guide RNAs into a cell, the cell's genome can be cut ata desired location, allowing existing genes to be removed and/or newones added [07].

According to preferred embodiment of the invention, a DNA end bindingprotein known as Gam is used to prevent the action of the DNA repairmachinery upon Cas9 cleavage. Gam is a protein from bacteriophage Muthat is orthologue to the Ku protein of NHEJ systems [44]. It is howevernot involved in repair but protects the Mu phage DNA in its linear formfrom host exonucleases [45]. Gam binds double strand ends (DSE) andprotects them from recBCD exonuclease activity. It was shown that uponUV exposure, the survival of cells expressing Gam is similar to that ofa recB mutant, indicating that Gam blocks DNA repair [46]. The inventorsshown here that Gam expression can be combined with Cas9 targeting toefficiently kill bacteria even when using weak guide RNAs that wouldotherwise be tolerated by the cell.

The fact that not all targets are able to kill E. coli means that itmight be difficult to use Cas9 as a reliable tool for genome editing oras a sequence-specific antimicrobial. In order to make Cas9 killing morereliable, the inventors investigated methods to prevent DNA repair whichcan restore Cas9's or other endonucleases' ability to kill a bacterium(e.g., E. coli) even when directed by a weak crRNA. The Gam protein ofphage Mu binds double stranded ends and protects the phage DNA fromdegradation by host exonucleases. The inventors cloned the Mu gam geneunder the control of a pBAD promoter and measured the transformationefficiency of pCas9 programmed either with a spacer that they previouslydescribed as weak (lacZ1) or with a stronger spacer (lacZ2).Surprisingly, transformation of pCas9::lacZ1 in the presence ofarabinose led to ˜250× fewer colonies than in the absence of arabinose,while the expression of Gam had no effect on CFU numbers of anon-targeting control pCas9 plasmid. Also surprisingly, the efficiencyof killing of the lacZ2 spacer, which is already good, was furtherimproved ˜14× in the presence of Gam. Together, these resultsdemonstrate the usefulness of using an inhibitor of double strand breakrepair pathways in combination with Cas9 or other endonucleases toensure that it will kill the targeted cells.

As used herein the term “plasmid” relates to a small DNA molecule withina cell that is physically separated from a chromosomal DNA and canreplicate independently. The plasmids are most commonly found inbacteria as small circular, double-stranded DNA molecules; however,plasmids are sometimes present in archaea and eukaryotic organisms. Theartificial plasmids are widely used as vectors in molecular cloning,serving to drive the replication of recombinant DNA sequences withinhost organisms.

As used herein the term “phagmid” refers to a plasmid that can bepackaged into a phage capsid. This includes f1/M13 filamentous phagesbut also other type of phages. A phagemid is thus defined as a DNAcircuit that can be packaged into a phage capsid and delivered to targetbacteria. Typically a phagemid is obtained from a temperate phage bycloning the packaging signal of the phage on a plasmid. The productionof phagemid particles, i.e. the plasmid DNA packaged into the phageprotein capsids, is achieved by using a production strain carrying thelysogenic helper phage and the phagemid. Upon induction of the phagelytic cycle, phage capsids are produced that will package the phagemidDNA. The packaging signal can be removed from the helper phage in orderto obtain pure phagemid particles.

According to one embodiment of the method of the present invention aphagemid(s) or bacterial conjugation can be used to deliver theendonuclease and the inhibitor of DSB repair, particularly a proteinthat binds to the ends of the double-stranded break and inhibits DSBrepair. Suitable phagemids can be based on the following phages,including M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1 Puna-like, P2,13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3phage, P37, MS2, Qβ, or Phi X 174. Preferred phages are selected from λphage, T2 phage, T4 phage, T7 phage, T12 phage, R17 phage, M13 phage,MS2 phage, G4 phage, P1 phage, Enterobacteria phage P2, P4 phage, Phi X174 phage, N4 phage, Pseudomonas phage ϕ6, ϕ29 phage, and 186 phage.Other suitable phages can be found in the Felix d'Herelle collection(http://www.phage.ulaval.ca/en/accueil/).

According to one embodiment of the invention, one phagimid or plasmidencodes the endonuclease and another phagemid or plasmid encodes theprotein inhibiting DSB repair.

According to another embodiment, the protein inhibiting DSB repair issynthetized prior to contacting it with bacterium.

In a specific embodiment of the method, the prokaryotic cell, inparticular a bacterial cell, is transformed with DNA polynucleotide(s)encoding the polypeptide(s) and RNA transcripts of a bacterialCRISPR-Cas system comprising (i) a nucleic acid molecule encoding aprogrammable double-stranded DNA Cas endonuclease and (ii) DNAmolecule(s) comprising a combination of sequences encoding a guide RNA(gRNA) encompassing the crRNA and tracrRNA transcripts, wherein the DNAmolecule(s) is (are) either a two-molecule DNA encoding crDNA andtracrRNA independently or a chimeric DNA encoding a singlecrRNA-tracrRNA transcript (said chimeric DNA being designated as sgRNAfor single guide RNA), wherein the nucleic acid molecule and DNAmolecule(s) are under the control of regulatory elements fortranscription including promoter(s).

The crRNA (CRISPR RNA) is encoded by a DNA molecule comprising a CRISPRarray that comprises one or multiple distinct DNA sequence(s)(designated spacer(s)) suitable for screening or for recognition of andbase pairing hydridization to one or respectively multiple distincttarget nucleotide sequence(s) in a genomic nucleic acid in saidprokaryotic cell said spacer sequence(s) being framed by a repeatsequence, said DNA being transcribed as a primary transcript which givesrise to short crRNA by processing.

crRNA is obtained as a result of the processing of the primarytranscript of the CRISPR array, said processing involving binding of thetracrRNA transcript to the repeat region of the CRISPR primarytranscript and recognition of the tracrRNA::CRISPR RNA duplex by Cas,especially Cas 9 and cleavage by the host RNAselII.

According to the invention, the DNA polynucleotide(s) encoding thepolypeptide(s) and RNA transcripts of the CRISPR-Cas system are borne bya vector, in particular a recombinant plasmid(s) or phagemid(s).

In the DNA polynucleotide(s) encoding the guide RNA, the DNA moleculeencoding the tracrRNA can be combined or fused, on a single plasmid orphagemid, with the sequence encoding the crRNA comprising the CRISPRarray. In the CRISPR array a leader sequence may be present adjacent tothe spacer sequences framed by the repeat sequences.

In the plasmid(s) or phagemid(s), the coding sequences are under thecontrol of a promoter for transcription, in particular a constitutivepromoter or an inducible promoter.

According to the invention, the DNA polynucleotide(s) encoding thepolypeptide(s) and RNA transcripts of the CRISPR-Cas system comprise(s)(i) a nucleic acid molecule encoding a programmable double-stranded DNACas endocuclease and (ii) DNA molecule(s) comprising a combination of oralternatively a fusion of a sequence encoding a guide RNA (gRNA) whichcomprises the crRNA and the tracrRNA transcripts, wherein the DNAmolecule encoding the crRNA encompasses (a) a CRISPR array and (b) asequence complementary to part of a sequence of the tracRNA codingsequence.

In a particular embodiment, the CRISPR system is from a Streptococcus,particularly a Streptococcus pyogenes.

In one embodiment, the bacterium is a Mycobacterium, in particularMycobacterium tuberculosis, or a Pseudomonas, in particular Pseudomonasaeruginosa. In various embodiments, the bacterium is selected from thegroup comprising or consisting of an E. coli, a Bacillus subtilis, aPseudomonas Aeruginosa, a Mycobacteria, a Streptococcus pyogenes, or aStaplylococcus aureus. In various embodiments, the bacterium is selectedfrom the group comprising or consisting of an Enterococci, Clostridiumdiffcile, Enterobacteriaceae, Neisseria gonorrhoeae, Acinetobacter,Campylobacter, Salmonella, Shigella, or Streptococcus pneumonia.

In preferred embodiment, bacteria are selected from the group comprisingEnterobacter, Streptococci, Staphylococci, Enterococci, particularly E.coli, Salmonella, Pseudomonas Aeruginosa, Mycobacterium tuberculosis,Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecali.

Particularly, bacteria are antibiotic resistant bacteria.

The invention further relates to the use of the method of the inventionfor making a bacterium more susceptible to an antibiotic comprisingcontacting the bacterium with an endonuclease, preferably encoded by arecombinant phagemid(s) or plasmid(s), wherein the endonuclease createsa double-stranded break in an antibiotic resistance gene encoded by thebacterium, the antibiotic, and an exogenous molecule that inhibits DNArepair. In a preferred embodiment, the molecule is an exogenous proteinthat binds to the ends of the double-stranded break and inhibits DSBrepair that binds to the ends of the double-stranded break and inhibitsDSB repair. Preferably, the protein is Mu phage Gam protein, a lambdaphage Gam protein, or a phage T7 gp5.9 protein. Preferably, the proteinis a recBCD or AddAB inhibitor. Other inhibitors of recBCD are forexample genes abc1 and abc2 from phage P22 [43].

Introduction of a DSB in the chromosome (and in the presence of Gam)will kill the bacterium, no matter where the target is. If the target isin an antibiotic resistance gene, the bacterium will die and will thusnot be resensitized to the antibiotic. On the other hand, if the targetis carried by a plasmid, no matter where the target is on the plasmidsequence, then the plasmid will be destroyed. If the plasmid carries anantibiotic resistance gene, then the bacterium will be made moresusceptible to the antibiotic.

Preferably, the double-strand break(s) is (are) performed in achromosomal context, i.e. on a double strand DNA when it is present onthe chromosomal DNA of the cell, either naturally or as a result ofinsertion of a DNA sequence in said cell chromosome(s).

The prokaryotic cell, in particular the bacterial cell used to carry outthe methods of the invention can be an isolated cell or a culture ofcells.

The invention also relates to a method for making a bacterium moresusceptible to an antibiotic comprising contacting the bacterium with anendonuclease, preferably encoded by a recombinant phagemid(s) orplasmid(s), wherein the endonuclease creates a double-stranded break inan antibiotic resistance gene encoded by the bacterium, the antibiotic,and an exogenous molecule that inhibits DNA repair. This method have thesame characteristics as the method of the invention for making abacterium more susceptible to an antibiotic described above.

The invention encompasses phagemid vectors and plasmids encodingendonucleases and/or proteins that inhibit DSB repair. Preferably, thephagemid or plasmid vector(s) encodes the endnuclease and the proteinthat binds to the ends of the double-stranded break and inhibits DSBrepair.

According to one embodiment of the invention, the plasmid or phagimigvector encodes only the endonuclease and the protein inhibiting DSBrepair is encoded by another plasmid or phagimid.

In one embodiment the endonuclease encoded by phagemid and/or plasmidvectors is selected from a meganuclease, preferably a Homingendonuclease (HEs) or an artificial endonuclease, preferably selectedfrom the group comprising a Zinc Finger Nuclease, TALEN and Cas nucleaseof CRISPR-Cas system, more preferably, a Cas9 nuclease, a guide RNA, andthe exogenous protein is selected from the group comprising Mu phage Gamprotein, a lambda phage Gam protein, a phage T7 gp5.9 protein,preferably a Mu phage Gam protein and a lambda phage Gam protein.

According to one preferred embodiment of the invention, the phagemid(s)or plasmid(s) encode a nuclease, a guide RNA, and an exogenous protein.

According to another preferred embodiment the guide RNA encompasses atwo molecule DNA encoding a CRISPR system's crRNA and tracrRNAindependently or a chimeric DNA (sgRNA) encoding a single crRNA-tracrRNAtranscript.

Most preferably, the phagemid(s) or plamid(s) encode a Cas9 nuclease, aguide RNA, and an exogenous Gam protein. In various embodiments, theguide RNA targets an antibiotic resistance plasmid or a plasmid carryingvirulence genes. In various embodiments, the guide RNA targets thebacterial chromosome. In various embodiments, the phagemid vector is aP1 bacteriophage. In various embodiments, the phagemid vector is a λbacteriophage.

The invention accordingly relates in particular to a plasmid or phagemidvector encoding a CRISPR-Cas system, in particular wherein theCRISPR-Cas system is a type II CRISPR associated (Cas) system comprisingDNA polynucleotide(s) encoding the polypeptide(s) and RNA transcripts ofa bacterial CRISPR-Cas system encompassing (i) a polynucleotidecomprising a sequence encoding a Cas double-stranded DNA endonuclease,in particular Cas 9, (ii) DNA molecule(s) comprising a combination ofsequences encoding a guide RNA (gRNA) encompassing the crRNA andtracrRNA transcripts, wherein the DNA molecule(s) is (are) either atwo-molecule DNA encoding crRNA and tracrRNA independently or a chimericDNA encoding a single crRNA-tracrRNA transcript (said chimeric DNA beingdesignated as sgRNA for single guide RNA), wherein the nucleic acidmolecule and DNA molecule(s) are under the control of regulatoryelements for transcription including promoter(s) wherein in the gRNA asuccession of DNA targeting nucleotide sequences (designated spacers)having 20 to 40 nucleotides, in particular 30 nucleotides or any valuein the ranges defined by the thus disclosed values is present andwherein each spacer's transcript is intended to screen or is able totarget a specific DNA sequence of interest to form a RNA-DNA interactionwith the target sequence and wherein each spacer is framed by identicalDNA repeat sequences. Said CRISPR associated Cas system is provided inthe cell as a single operon or as multiple polynucleotides.

The so-called spacer sequence may be designed to target a specificnucleotide sequence in the chromosomal DNA of the cell, i.e. to target adetermined polynucleotide strand. In a particular embodiment, the spacersequence may be designed to possibly hybridize with a known sequence ofnucleotides of a chromosome in a determined polynucleotide of interest.Alternatively it may be designed randomly, i.e., with no predeterminedtarget in the chromosomal sequence of the cell and accordingly thepolynucleotide of interest may be a sequence randomly targeted orscreened in said chromosomal DNA. The spacer(s) sequence may thus be thenatural sequence of the CRISPR system or may be a sequence heterologousto said natural CRISPR system, selected for its ability to target aproper determined or undetermined sequence in the chromosomal DNA of theprokaryotic cell. Accordingly, the CRISPR system is designed forprogrammed targeting in the chromosomal DNA of the prokaryotic cellwhether the sequence of the targeted polynucleotide comprising thetarget is known or not said sequence being of prokaryotic origin orbrought to the prokaryotic cell from a eukaryotic DNA by recombinationof the prokaryotic cell.

The targeted polynucleotide may be of any type and is further disclosedhereafter.

The “repeat sequence” that frames the spacer sequences in the CRISPRsystem is involved in the maturation of the preCRISPR RNA transcript andin the mature transcript designated crRNA. Accordingly, part of therepeat sequence is contained in the crRNA. The repeat sequence mayencompass 20 to 50, in particular 20 to 40 or 35 to 40 nucleotides orany range that may be defined having recourse to these disclosed values,or any value in-between and especially 36 nucleotides as illustrated inthe example of S. pyrogenes.

For Illustration, particular repeat sequences are SEQ ID Nos 1 to 10below (these sequences correspond to the SEQ ID Nos: 99 to 108 of thepriority application):

SEQ ID No. 1 (GTTTTTGTACTCTCAAGATTTAAGTAACTGTACAAC) SEQ ID No. 2(GATATAAACCTAATTACCTCGAGAGGGGACGGAAAC) SEQ ID No. 3(GTTTTGGAACCATTCGAAACAACACAGCTCTAAAAC) SEQ ID No. 4(GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC) SEQ ID No. 5(ATTTCAATCCACTCACCCATGAAGGGTGAGAC) SEQ ID No. 6(GTTTCAGTAGCTAGATTATTTGATATACTGCTGTTAG) SEQ ID No. 7(AATCAGAGAATACCCCGTATAAAAGGGGACGAGAAC) SEQ ID No. 8(GTTCACTGCCGCACAGGCAGCTTAGAAA) SEQ ID No. 9(GGTTGTAGCTCCCTTTCTCATTTCGCAGTGCTACAAT) SEQ ID No. 10(CCGGATTCCCGCCTGCGCGGGAATGACG)

As mentioned above, alternatively to being composed of a DNA moleculeencoding the Cas9 protein and DNA molecules encoding tracrRNA and crRNAtranscripts provided as separate genes, the CRISPR-Cas system is a typeII CRISPR associated (Cas) system encompassing (i) a polynucleotidecomprising a sequence encoding a Cas double-stranded DNA endonuclease,in particular Cas 9, and (ii) a chimeric DNA that is transcribed as achimeric RNA i.e., single guide RNA (sgRNA) encompassing a fusion of thenucleic acids transcribed as the tracrRNA and the crRNA on the same oron a different plasmid or phagemid as the one expressing Cas.

The CRISPR associated system may encompass a Cas double-stranded DNAendonuclease the gene of which flanks the polynucleotide encoding thegRNA or the sgRNA in a Cas operon. This CRISPR associated system mayinvolve in particular the programmable endonuclease Cas 9 as describedin detail in the Examples and illustrated for the performance of DSB inE. coli.

Alternatively, the gene of the CAS endonuclease may be provided on aseparate DNA construct. The polynucleotide encoding the gRNA or thesgRNA (CRISPR genetic construct) and the polynucleotide encoding theendonuclease may thus be introduced into the cell by transformation witha single or multiple plasmids or phagemids.

The CRISPR array comprises one or multiple spacer sequences framed by arepeat sequence that are transcribed into pre-CRISPR RNA which isprocessed to small RNA sequences (crRNA) that allow DNA targeting in thechromosomal nucleic acid of the prokaryotic cell, the DNA target beingcomplementary enough to the spacer transcript present in the crRNA tohybridize with it when the target DNA comprises, in addition,immediately downstream to the target region, a recognition sequencedesignated PAM sequence (Photospacer-adjacent Motif).

The spacer sequence(s) of the CRISPR array may advantageously consist of20 to 40 nucleotides, in particular 30 nucleotides or any value in theranges defined by the thus disclosed values and the sequence(s) arechosen by reference to the target in the chromosomal nucleic acid or asa random sequence when no specific sequence is targeted in the nucleicacid. The repeat sequence in the CRISPR system is one which may beprocessed by the enzymes of the prokaryotic cell thereby giving rise tothe small crRNA encompassing a transcript of at least part of the repeatsequence. Illustration of spacer sequences is provided herein as SEQ IDNo. 1 to 10 and in the Examples.

The polynucleotide transcribed into the tracrRNA is a short RNAantisense to the precursor RNA. The formed tracrRNA enables the loadingof the crRNA on the Cas protein and accordingly participates in aRNA-protein complex that involves tracrRNA, crRNA and Cas protein(so-called dual-RNA:Cas) that targets the chromosomal nucleic acid tothen allow the DSB to take place at the targeted loci. As mentionedabove, the nucleic acids transcribed as the tracrRNA and the crRNA maybe fused in a chimeric nucleic acid giving rise to a sgRNA when theCRISPR system is active in the cell.

In a particular embodiment, the CRISPR-Cas system is composed ofassociated nucleic acid molecules, one of them encoding the Cas 9protein and the additional one(s) being transcribed as the tracrRNA, andas the crRNA, the nucleic acids being under the control of distinct orcommon regulatory sequences for expression, including a promoter. In aparticular embodiment the tracrRNA and crRNA give rise to a chimerictranscript i.e., a sgRNA and are under the control of the sametranscription promoter.

Optionally, the nucleic acid molecules are borne by different plasmidsor phagemids and remain independent. The polynucleotide or nucleic acidmolecules are under the control of suitable transcription or expressioncontrol elements.

In a particular embodiment, the CRISPR-associated Cas9 system is encodedby a nucleic acid from a Streptococcus genus in particular from aStreptococcus pyrogenes strain.

In a preferred embodiment, the CRISPR system comprises the sequence ofthe leader and the repeat sequence from the locus of Streptococcuspyrogenes disclosed as SF370 under accession number NC_002737.

In a particular embodiment the CRISPR-Cas system is provided by plasmidpCas9 (also named pCas9-a) having the sequence of SEQ ID NO: 60(indicated as SEQ ID No. 117 in the priority application) or aderivative thereof, particularly a phagemid, wherein the regioncorresponding to the control spacer, from nucleotide position 6520 toposition 6549, is substituted by one or multiple spacer(s) of choice oris provided by plasmid pCas9-LacZ2 having the sequence of SEQ ID NO: 61(indicated as SEQ ID No. 119 in the priority application) or aderivative thereof, particularly a phagemid, wherein the region fromnucleotide position 6520 to position 6549 (CRISPR target ELZ2) issubstituted by one or multiple spacer(s) of choice.

Other bacterial species may provide the Cas 9 protein or nucleic acidmolecule encoding the Cas 9 protein. These species include, forillustrative purposes only: Francisella novicida, Legionellapneumophila, Streptococcus thermophulus, Streptococcus mutans,Coriobacterium glomerans, Staphylococcus lugdumensis, Enterococcusfaecalis, Mycoplasma canis, Campylobacter jejuni, Neisseriameningitidis, Pasteurella multocida.

According to another particular embodiment of the invention, the CRISPRsystem is provided by two plasmids or phagemids used for thetransformation of the cell: a first plasmid or phagemid provides thepolynucleotides encoding the Cas protein (said first plasmid or phagemidcan be built on the same basis as the pCas9 provided it is notrecombined with the sequence encoding the crRNA and the tracrRNAtranscripts), a second plasmid or phagemid that encodes the crRNA andthe tracrRNA transcripts said second plasmid or phagemid comprising in aparticular embodiment a DNA polynucleotide that comprises the “gRNAscaffold for the CRISPR/Cas 9 system” having the sequence fromnucleotide 1565 to nucleotide 1640 in the sequence of SEQ ID NO:62(indicated as SEQ ID No. 123.in the priority application).

Said second plasmid or phagemid can be in particular derived fromplasmid psgRNAc BsaI (SEQ ID No. 62).

According to a particular embodiment of the invention, in the secondplasmid or phagemid, the DNA polynucleotide(s) comprise(s) in addition,the sequence of the tracrRNA ending at position 1647 in the sequence ofSEQ ID No. 62.

According to a particular embodiment of the invention, said secondrecombinant plasmid or phagemid encoding the single guide RNA for theCRISPR/Cas 9 system comprises the sequence of SEQ ID No.62. In saidphagemid, the sequence of the control spacer from nucleotide position1545 to nucleotide position 1564 in the sequence of SEQ ID No. 62 may besubstituted by any selected sequence of choice for a spacer and inparticular a spacer sequence disclosed herein.

The protein that binds to the ends of the double-stranded break andinhibits DSB repair can be expressed from either the first or secondrecombinant plasmid or phagemid or on a third plasmid or phagemid.

In a particular embodiment the CRISPR-associated Cas9 system isexpressed in the recombinant prokaryotic cell as a ternary complex thatinvolves tracrRNA paired to crRNA and bound to Cas9 wherein the crRNAtargets DNA on the chromosome of the recombinant prokaryotic cell tocause at least one DSB in the DNA.

In a particular embodiment, the CRISPR array comprises 1 to 10, inparticular 1 to 5 spacer sequences. When multiple spacer sequences arethus contained in the CRISPR array, this array is transcribed asmultiple crRNA molecules having distinct spacer sequence, therebyenabling multiplex DSB to take place at different loci of thechromosomal DNA of the prokaryotic cell.

In a particular embodiment of the invention, the method is used tointroduce DSBs at any locus (loci) of interest in the chromosome simplyby changing the sequence of the guide spacer.

According to the invention, a chromosomal sequence, in which a DSB isgenerated is defined as a “polynucleotide of interest”. According to aparticular embodiment, as stated above, a polynucleotide of interest canbe a targeted polynucleotide despite it does not require that itsnucleotide sequence upstream and downstream of the cut site for DSB isdetermined. Targeting in this respect may rely on criteria such aslocation into the chromosome, functional parameters of the target DNA,which are known or are to be identified, involvement in phenotypictraits, or structural parameters of the DNA. Targeting may take intoconsideration possible functional or structural relationship amongmultiple DNA. Alternatively, in another embodiment of the invention, thesaid polynucleotide of interest is a nucleic acid which is heterologouswith respect to the natural chromosomal nucleic acid of the prokaryoticcells wherein the invention is carried out. The expression“heterologous” means that said nucleic acid is originating from adifferent cell, species or organism than the cell type which is used toperform the invention, or is a non-naturally occurring nucleic acid suchas a chimeric or an artificial nucleic acid. Such heterologouspolynucleotide may nevertheless have been inserted in the genome of thecell, possibly using recombinant technologies. In a particularembodiment the heterologous sequence may be a eukaryotic DNA sequence,especially a chromosomal eukaryotic sequence.

The polynucleotide of interest may comprise the cleavage site where theDSB is generated and the required PAM (photospacer adjacent motif)sequence the latter corresponding to a sequence either naturally presentin the target DNA or introduced in it. The PAM sequence is recognized bythe Cas protein and is accordingly dependent of the choice of thisprotein. The PAM sequence functional with the Cas9 protein is a sequence5′XGG3′ on the complementary strand of the target polynucleotide,wherein X means any nucleotide.

Alternatively, the polynucleotide of interest may have been insertedinto the chromosomal substrate through the action of an agent or of anorganism, such as a bactreiophage.

The polynucleotide of interest can be in its native form, or it may haveundergone modifications with respect to a reference wild-type form ifany, especially when it is a polynucleotide which is inserted andintegrated in the chromosomes of the cell. The modifications may becarried out prior to or after the insertion into the cell or as a resultof recombination into the cell genome.

The polynucleotide of interest of the invention, either known in itscomposition or randomly selected (random polynucleotide), may be anucleic acid of a gene or of a gene fragment, including an exon, anintron, an expression regulatory sequence such as a promoter, a codingsequence, a non-coding sequence. It may be a nucleic acid of prokaryoticor of eukaryotic origin. It may be a nucleic acid, especially ofprokaryotic origin, originating from a pathogenic organism, such as aviral or bacterial or parasite nucleic acid, including a protein codingsequence. It may be a nucleic acid of prokaryotic origin, originatingfrom a non-pathogenic organism.

The polynucleotide of interest of the invention may be present as asingle sequence in the chromosomal substrate of the cell or rather maybe present as multiple copies of its sequence, either contiguous in thechromosome or spread on the chromosome. In a particular embodiment,different polynucleotides, i.e., polynucleotides having differentnucleotide sequences, present in the chromosomal substrate of the cellare subject to the double-strand break.

According to a first step of the method of the invention, a DSB isgenerated in a targeted way in the DNA sequence of the targetedpolynucleotide, which means that a specific locus of the polynucleotideis the target of the break in the prokaryotic cell.

In another embodiment of the invention, the site for the DSB is not asingle site, i.e., there can be multiple sites in the polynucleotide.

Double-strand break site for the purpose of the invention may be uniquein the polynucleotide of interest (giving rise to a single DSB event) ormay be multiple (giving rise to multiple DSB events) especially as aresult of the presence of multiple distinct spacers in the CRISPRsystem. DSB sites are indeed determined by the sequence of the spacer(s)of the CRISPR system and the presence in the chromosomal DNA (possiblyafter modification) of PAM sequences.

As a result of the CRISPR construct used, it is possible to performdouble-strand break, especially targeted DSB, in one or more than onelocus of the chromosomal DNA of prokaryotic cells.

As examples of DNA targets of interest, the invention provides nucleicacids consisting in or contained in:

-   -   a gene expressing an enzyme, such as a kinase, in particular        wherein the sequence of the polynucleotide of interest encodes        the active site of the enzyme,    -   a gene expressing a cell receptor,    -   a gene expressing a structural protein, a secreted protein,    -   a gene expressing resistance to an antibiotic, or to a drug in        general    -   a gene expressing a toxic protein or a toxic factor,    -   a gene expressing a virulence protein or a virulence factor,    -   a polynucleotide, especially a gene of a pathogen such as a        virus a bacterium or a parasite,    -   regulatory sequences for transcription or for expression of said        genes.

In one embodiment, the method of the invention may be used forincreasing the nuclease activity, particularly when in suboptimalconditions (variating the in vitro used conditions) or when there is oneor several mutations in target DNA, the nuclease activity is decreased.Thus, the method of the invention may be used for enhancing nucleaseefficiency.

In one aspect the present invention also relates to a host cellcomprising a vector encoding an endonuclease according to the inventionand a vector encoding a protein inhibiting DSB repair.

In one embodiment, the host cell can contain a vector encoding anendonuclease and a protein inhibiting DSB repair.

Such host cell may be used for research purposes.

In another aspect, the invention also relates to a pharmaceuticalcomposition comprising the vector as described above and apharmaceutical acceptable vehicle for the treatment of diseases due to abacterium infection.

In the context of the present invention “pharmaceutical acceptablevehicle” refers to a compound, or a combination of compounds, entering apharmaceutical composition that does not cause secondary reactions andthat, for example, facilitates administration of the active compounds,increases its lifespan and/or effectiveness in the organism, increasesits solubility in solution or improves its storage. Such pharmaceuticalcarriers are well-known and will be adapted by a person skilled in theart according to the nature and the administration route of the activecompounds selected.

The pharmaceutical composition according to the invention furthercomprises a vector encoding the protein inhibiting DSB repair or proteininhibiting DSB repair.

In one embodiment, the pharmaceutical composition is suitable for thetreatment of diseases due to a bacterium selected from the groupcomprising Enterobacter, Streptococci, Staphylococci, Enterococci,Salmonella, Pseudomonas, Mycobacterium.

In another embodiment, the pharmaceutical composition further comprisingan antibiotic, particularly a suitable antibiotic for treating infectiondue to a bacterium selected from the group of Enterobacter,Streptococci, Staphylococci, Enterococci Salmonella, Pseudomonas,Mycobacterium.

According to a further aspect, the invention relates to a productcomprising

at least one phagemid or plasmid vector of the invention as describedabove or a pharmaceutical composition of the invention, and

at least another therapeutic agent, in particular an antibiotic

as a combination product for simultaneous, separate or sequential usefor the treatment of at least one disease due to a bacterium infection,particularly an infection due to at least one bacterium selected fromthe group comprising Enterobacter, Streptococci, Staphylococci,Enterococci, Salmonella, Pseudomonas, Mycobacterium.

According to another aspect, the invention also relates to a method fortreating diseases due to a bacterial infection, said method comprisingadministering at least one phagemid or plasmid vector or apharmaceutical composition or a product according to the invention to asubject suffering from a bacterium infection.

-   -   According to one embodiment, the therapeutic method of the        invention is used for treating a patient suffering from an        infection with at least one bacterium selected in the group        comprising Enterobacter, Streptococci, Staphylococci,        Enterococci, Salmonella, Pseudomonas, Mycobacterium.

Further characteristics and embodiments will be apparent from theExamples which follow and from the figures.

EXAMPLES Example 1 Effect of Double Strand Breaks Introduced by Cas9 onCell Death and Conditions for Survival to Such DNA Damage

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) andCRISPR associated (Cas) genes are the adaptive immune system of bacteriaand archaea [1]. The RNA-guided Cas9 nuclease from Streptococcuspyogenes has emerged as a useful and versatile tool [2]. The ease withwhich it can be reprogrammed has in particular been driving its adoptionfor genome editing applications. Cas9 is guided by a small CRISPR RNA(crRNA) that is processed from the initial transcript of the CRISPRlocus by Cas9 together with a trans-activating CRISPR RNA (tracrRNA) andthe host RNAselII [3]. Both the tracrRNA and the processed crRNA remainbound to Cas9 and act as a complex to direct interference against targetDNA molecules[4]. Alternatively, the crRNA and tracrRNA can be fusedforming a chimeric single guide RNA (sgRNA) [4]. Cas9 scans DNA lookingfor a short sequence motif known as the Protospacer Adjacent Motif (PAM)[5]. Once a PAM is found, DNA is unwound to make base-pair contactsbetween the crRNA and the target DNA. If base-pairing occurs, aconformational shift in Cas9 brings two nuclease domains in contact withthe target DNA leading to the creation of a double strand break (DSB)[6].

Genome editing using Cas9 has been reported in a large number ofeukaryotes including insects, plants, mammals, yeast, zebrafish, xenopusand nematode [2]. However it has so far only been demonstrated in a fewbacteria species and with a handful of target positions [7-9]. Ineukaryotic cells DSB introduced by Cas9 can be repaired through HomologyDirected Repair (HDR) with a template DNA molecule carrying a mutationof interest [10,11]. Alternatively, error-prone repair by Non-HomologousEnd Joining (NHEJ) can lead to small indels at the target site which areused to knockout genes [10,11]. In contrast, most bacteria lack a NHEJsystem [12,13] and Cas9-induced breaks in bacterial genomes lead to celldeath [14-16]. This repair pathway thus cannot be used to introducesmall deletions and knockout genes. However, the ability to killbacteria carrying a specific sequence in the chromosome can be used inconjunction with a mutagenesis strategy to select for cells that carry adesired mutation [7].

More recently, the ability of chromosome-targeting CRISPR systems tokill bacteria was used to develop sequence-specific antimicrobials[14,15]. In these studies phage capsids are used to deliver a CRISPRsystem programmed to target antibiotic resistance or virulence geneseither in E. coli or S. aureus. In both cases this strategy was able toefficiently kill the targeted bacteria specifically.

First, the inventors investigated why DSB introduced by Cas9 leads tocell death and whether some cells can survive such DNA damage.

Example 2 Bacterial Strains and Media

E. coli strains were grown in Luria-Bertani (LB) broth (10 g Tryptone, 5g Yeast Extract, 10 g NaCl, add ddH2O to 1000 ml, PH7.5, autoclaved).1.5% LB Agar was used as solid medium. Different antibiotics (20 ug/mlchloramphenicol, 100 ug/ml carbenicillin, 50 ug/ml kanamycin) were usedas needed. Plates containing IPTG (100 uM) and X-gal (40 ug/ml) wereused for blue/white screening. Escherichia coli strain MG1656 (aΔlacI-lacZ derivative of MG1655) was used as a cloning strain forplasmid pCas9::lacZ2 (see below). E. coli strains N4278 (MG1655recB268::Tn10)²⁹, MG1655 RecA::Tn10 and JJC443 (lexAind3 MalF::Tn10)³⁰are gifts from the Mazel lab.

Example 3 Plasmid Cloning

pCRRNA was assembled by amplification of pCRISPR using primer B299/LC34and of the tracrRNA fragment from pCas9 using primers LC35/LC36,followed by Gibson assembly [31]. Novel spacers were cloned into pCRRNAor pCas9 plasmids as previously described [7]. The vector was digestedwith BsaI, followed by ligation of annealed oligonucleotides designed asfollows: 5′-aaac+(target sequence)+g-3′ and 5′-aaaac+(reverse complementof the target sequence)-3′. A list of all spacers tested in this studyis provided in (Table 2 in the present application was indicated withthe number 4 in the text of the priority application corresponding tothe table 2 of the priority application).

The pLCX plasmid was assembled from the pCRISPR backbone amplified usingprimers LC41/LC42 and two lacZ fragments amplified from MG1655 genomicDNA using primers LC38/LC39 and LC37/LC40. The pZA31-sulA-GFP plasmidwas assembled from pZA31-Luc [32] linearized with primers LC192/LC193,the sulA promoter fragment amplified with primers LC194/LC196 andGFPmut2 [33] amplified with primers LC191/LC195. All PCR primers arelisted in (Table 3 in the present application was indicated with thenumber 5 in the text of the priority application corresponding to thetable 3 of the priority application).

Example 4 CRISPR Transformation Assays

The pCRRNA or pCas9 plasmids (with different spacers) were transformedin recipient E. coli strains by chemical transformation using 100 ng ofplasmid DNA. CFU numbers were normalized by pUC19 transformationefficiency. All transformations were repeated at least 3 times.

Example 5 SOS Response

The pZA31-sulA-GFP plasmid was used to monitor SOS induction [34]. TheOSIP system [36] was used to integrate cas9 or dcas9 under the controlof a ptet promoter [20] in the chromosome of strains MG1655, N4278(MG1655 recB268:Tn10) [29], MG1655 RecA::Tn10 and JJC443 (lexAind3MalF::Tn10) [30] (Table 1 in the present application was indicated withthe number 3 in the text of the priority application corresponding tothe table 1 of the priority application). pCRRNA plasmids with differentspacers were transformed by chemical transformation. Colonies isolatedfrom the transformation plate were re-suspended in 200 ul LB in a 96well microtiter plate. The microtiter plate was loaded into a TECANinfinite M200 Pro machine. OD (600 nm) and GFP fluorescence (excitationfilter set to 486 nm and emission filter set to 518 nm) were measuredover a 10 hour time course. GFP values at OD of 0.4 are reported.

Example 6 Cloning of the pLC13 Plasmid

The pLC13 plasmid was constructed through Gibson assembly of plasmidpBAD18 [47, amplified with primers LC2/LC296 together with the gam geneof bacteriophage Mu amplified with primers LC397/LC398 from the genomicDNA of E. coli S17-1 LPIR[5]. The sequence of pLC13 (which is fullypresent in the text of the description of the priority application)corresponds to SEQ ID NO: 11 of the sequence listed annexed to thepresent specification.

Example 7 pCas9 Transformation and Plating Assay

The pCas9, pCas9:LacZ1 and pCas9:LacZ2 plasmids were transferred intoMG1655 cells carrying the pLC13 plasmid. Cells were plated on LB-agarwith or without 0.2% L-arabinose. Serial dilutions were performed toquantify CFU for each transformation.

TABLE 4 Primers used for pCas9 transfection. SEQ ID Primer NO: NamePrimer sequences (5′ to 3′) 12 LC2 CCTTCTTAAAGTTACCGAGCTCGAATTCGC 13LC296 TATATTTTAGGAATTCTAAAGATCTTTGACA GCTAGCTCAGTCCTAGGTATAATACTAGT 14LC397 ATCCGCCAAAACAGCCAATTAAATACCGGCT TCCTGTTC 15 LC398GCGAATTCGAGCTCGGTAACTTTAAGAAGGA GATATACCATGGCTAAACCAGCAAAACGT

Example 8 E. coli can Survive Cas9 Cleavage Through Homology DirectedRepair

Evidence that CRISPR interference directed against the chromosome leadsto cell death first came from the observation that an active CRISPRsystem and its target cannot coexist in the same cell [16-18].Transformation of E. coli by a plasmid carrying a CRISPR systemtargeting the chromosome is very inefficient, typically resulting in1,000-fold decrease in transformation efficiency compared to anon-targeting control [7,17, 19]. In a previous study, we took advantageof this to introduce a mutation in the rpsL gene of E. coli [7].Targeting of the rpsL gene by Cas9 killed the cells that did notincorporate a desired mutation provided by an oligonucleotide. Toinvestigate whether this approach could be extended to other loci, weprogrammed a plasmid-born CRISPR array to target 12 positions spreadthroughout the E. coli chromosome and compared them with the rpsL targetpreviously published. All targets were chosen in non-essential genes toensure that killing by Cas9 would be the result of DNA cleavage and notrepression of the target gene [20,21]. The pCRRNA plasmid carries thetracrRNA and a minimal CRISPR array consisting of the leader sequenceand a single spacer framed by two repeats. This plasmid was transformedin cells containing the pCas9 plasmid expressing Cas9 constitutively[7]. Surprisingly, 8 out of 12 spacers could be readily transformed withefficiencies comparable to that of the non-targeting control (FIG. 1).Interestingly, three of them (lacZ1, tsuB and wcaH) resulted in coloniessmaller than the control (FIG. 2). The inventors hypothesized that Cas9cleavage in these cells might be inefficient and that competition withthe bacteria repair system would stress the cells and slow down colonygrowth. To test this idea, the inventors repeated this transformationexperiment in cells deleted for recA. Consistently with inventors'hypothesis, no colonies could be recovered after transformation ofspacers lacZ1, tsuB and wcaH, but also after transformation of all theother spacers. This shows that all spacers are able to direct Cas9cleavage in the chromosome, including those that can be transformedefficiently, and all spacers induce lethal DSB in the absence of recA.However, only some spacers are able to kill cells in the presence ofrecA. This indicates that weak spacers might be tolerated in wild-typecells thanks to the Homology Directed Repair (HDR) pathway.

Homologous recombination can only rescue a DSB if an intact sisterchromosome is available. This suggests that for some spacers Cas9cleavage is not efficient enough to cut all copies of the chromosomesimultaneously. A corollary is that spacers that do lead to cell deathprobably kill the cells because no repair template is available. If thisis true, then providing an intact repair template during targetingshould be able to rescue the cells. To test this hypothesis theinventors constructed a plasmid, pLCX, carrying a 1 kb fragmenthomologous to the target region of spacer lacZ2, but with a pointmutation in the PAM motif blocking CRISPR interference (FIG. 1C).Transformation of the lacZ2 spacer led to ˜100× more colonies in thepresence of pLCX than in cells carrying a control empty plasmid, and nocolonies could be recovered in the recA mutant (FIG. 1D). The lacZ geneof the recovered colonies was sequenced and confirmed to carry the pointmutation provided by the pLCX plasmid, showing that it was indeed usedas a template for HDR.

Example 9 Cas9 Cleavage Leads to SOS Induction

Spacers that can be tolerated likely result in constant Cas9 cleavageand recA mediated repair. This should lead to an elevated level of SOSinduction. To test this the inventors integrated cas9 in the chromosomeunder the control of a ptet promoter and monitored SOS levels with a GFPreporter plasmid. Spacers were provided on the pCRRNA. Targeting withthe lacZ1 spacer led to elevated GFP fluorescence levels when aTc wasadded to the media, but more surprisingly also in the absence ofinduction (FIG. 3A and FIG. 4). This demonstrates that the ptet promotercontrolling Cas9 is leaky and that the small amount of Cas9 proteinsproduced can already lead to the introduction of DSB resulting in SOSinduction. Consistently with an induction of the SOS pathway, nofluorescence could be observed in recA, recB or lexA(ind−) mutants (FIG.3A). Mutations in the catalytic sites of Cas9 also abolished SOSinduction showing that cleavage of DNA and not mere binding is the causeof the SOS induction (FIG. 3A, dCas9). We further measured the SOSresponse triggered by all 13 spacers (FIG. 3B). Interestingly, thestrength of SOS induction correlates well with the ability of thespacers to kill the cells. This corroborates the idea that efficientcleavage of all copies of the chromosome is responsible for cell death.

TABLE 1 Integrated E. coli strains. This table shows the backbones andfragments used for integrations in the chromosome of E. coli followingmethods described previously (ref 35). The pOSIP backbone was removedfrom the chromosome using plasmid pE-FLP. Primers and templates used togenerate the fragments are listed in Table 3. Name of the new pOSIPIntegration Original pOSIP Strain strain Backbone Fragment 1 Fragment 2site strains backbone description LC-E01 pOSIP-KH Mt-LigD Mt-LigD HK022attB MG1655, removed MG1655 with promoter fragment RecB- Mt-LigD LC-E02pOSIP-KO Tet-dCas9 N/A 186 attB MG1655 removed MG1655 with inducibledCs9 LC-E03 pOSIP-KO Tet-wtCas9 N/A 186 attB MG1655 removed MG1655 withinducible wtCs9 LC-E05 pOSIP-KO Mt-Ku Mt-Ku 186 attB LC-E01 removedMG1655 with promoter fragment Mt-LigD and Mt-Ku LC-E06 pOSIP-KOTet-wtCas9 N/A 186 attB MG1655, removed MG1655 (RecA-) RecA- withinducible wtCs9 LC-E07 pOSIP-KO Tet-wtCas9 N/A 186 attB N4278 removedMG1655 (RecB-) with inducible wtCs9 LC-E08 pOSIP-KO Tet-wtCas9 N/A 186attB JJC443 removed MG1655 (LexA-) with inducible wtCs9

TABLE 2 CRISPR spacers used in this invention. CRISPR spacerCRISPR spacer sequence (from 5′ to 3′) / Targeted name SEQ ID NO: strandPAM lacZ1 TCACTGGCCGTCGTTTTACAACGTCGTGAC 16 Template TGG strand lacZ2CCATTACGGTCAATCCGCCGTTTGTTCCCA 17 Template CGG strand rpsLTACTTTACGCAGCGCGGAGTTCGGTTTTTT 18 Non template AGG strand mhpRGGAATTAATCGAAATGTTAGCCTCCCGCCC 19 Template CGG strand tsuBTAAGGTCTTCGTTCAGGGCATAGACCTTAA 20 Non template TGG strand wcaHTTTTCTCGCTGAGAAGCGTACCGGAGTACC 21 Template CGG strand irhAATTCCGCTGCGCAGTACCAGTGTGTTGGCG 22 Non template AGG strand eamBCAGCGGTACACCTTTTGAGTTGGGCGGGGG 23 Template CGG strand speAAGCAGAACGTCTGAATGTCGTTCCTCGTCT 24 Template GGG strand garDCGTGGTGGGGCTGAATCATTTGTACGGTTG 25 Template TGG strand treFGTACCGCGATTTACGCGCGGGGGCGGCCTC 26 Template CGG strand yfaPATTCGTGCACGTTTACGGCTGGTTCTCTCG 27 Template TGG strand adaGGTGCGTTACGCGCTGGCTGATTGTGAGCT 28 Template GGG strandThe SEQ ID Nos: 16 to 28 in table 2 correspond to SEQ ID Nos: 39 to 51of the priority application.

TABLE 3 Primers used in this invention. Fragments generated (of Primerprimer Name Primer sequences (from 5′ to 3′) SEQ ID NO: Templatefunction) B299 CATGAATTCAACTCAACAAGTCTCAGTGTGCTG 29 pCRISPR pCRISPRbackbone LC34 TTTAGGCGCTGCCATCTTAAGACGAAAGGGCCTCGTGATA 30 pCRISPRpCRISPR backbone LC35 TTCAGCACACTGAGACTTGTTGAGTTGAATTCATGAGTATT 31 pCas9TracrRNA AAGTATTGTTTTATGGCTGATA fragment LC36TATCACGAGGCCCTTTCGTCTTAAGATGGCAGCGCCTAAA 32 pCas9 TracrRNA fragment LC41TGCAGCGCGATCGTAATCAGGATCCCATGGTACGCGT 33 pCRISPR pCRISPR backbone LC42ACAGAACTTAATGGGCCCGAAGACGAAAGGGCCTCGT 34 pCRISPR pCRISPR backbone LC37TCCGCCGTTTGTTCCCACGTAGAATCCGACGGGTTGTTAC 35 MG1655 the 2nd lacZ genomichomologous DNA fragment LC38 GTAACAACCCGTCGGATTCTACGTGGGAACAAACGGCGGA 36MG1655 the 1st lacZ genomic homologous DNA fragment LC39ACGAGGCCCTTTCGTCTTCGGGCCCATTAAGTTCTGT 37 MG1655 the 1st lacZ genomichomologous DNA fragment LC40 ACGCGTACCATGGGATCCTGATTACGATCGCGCTGCA 38MG1655 the 2nd lacZ genomic homologous DNA fragment LC191GTCTAGGGCGGCGGATTTG 39 pDB127 GFPmut2 fragment LC192CGCTCTCCTGAGTAGGACAAAT 40 pZA31-Luc pbZA31-Luc backbone LC193ACAATTGAATACCGATCGGCCTCGTGATACGCCTAT 41 pZA31 -Luc pbZA31-Luc backboneLC194 ATAGGCGTATCACGAGGCCGATCGGTATTCAATTGTGCCCAA 42 MG1655 sulA promotergenomic fragment DNA LC195 CAGGGGCTGGATTGATTATGAGTAAAGGAGAAGAACTTTTC 43pDB127 GFPmut2 fragment LC196 TTCTTCTCCTTTACTCATAATCAATCCAGCCCCTGTGA 44MG1655 sulA promoter genomic fragment DNA LC95CTCCGACGCCGAACCCATACAACCTCCTTAGTACATCAAGCA 45 pE-FLP Mt-LigD promoterLC96 GCAGGACGCCCGCCATAAACTGCCAGGAATTGGGGATCGGG 46 pE-FLP Mt-LigDGGGTTCCGCGCACATTT promoter or Mt-Ku promoter LC94TGCTTGATGTACTAAGGAGGTTGTATGGGTTCGGCGTCGGAG 47 M. Mt-LigD tuberculosisfragment H37Rv genomic DNA LC98AGTTTAGGTTAGGCGCCATGCATCTCGAGGCATGCCTGCATC 48 M. Mt-LigDATTCGCGCACCACCTCA tuberculosis fragment H37Rv genomic DNA LC93CGTCCAAATGGCTCGCATACAACCTCCTTAGTACATCAAGCA 49 pE-FLP Mt-Ku promoter LC92TGCTTGATGTACTAAGGAGGTTGTATGCGAGCCATTTGGACG 50 M. Mt-Ku tuberculosisfragment H37Rv genomic DNA LC97AGTTTAGGTTAGGCGCCATGCATCTCGAGGCATGCCTGCATC 51 M. Mt-Ku ACGGAGGCGTTGGGACtuberculosis fragment H37Rv genomic DNA LC100GCAGGACGCCCGCCATAAACTGCCAGGAATTGGGGATCGGT 52 pdCas9- Tet-dCas9 orTAAGACCCACTTTCACATTTAAG bacteria or Tet-Cas9 pwtCas9- fragment bacteriaLC101 AGTTTAGGTTAGGCGCCATGCATCTCGAGGCATGCCTGC 53 pdCas9- Tet-dCas9 orATATAAACGCAGAAAGGCCC bacteria or Tet-Cas9 pwtCas9- fragment bacteriaLC33 GACTGGAAAGCGGGCAGT 54 Sequencing LC47 CGCACGATAGAGATTCGGGA 55Sequencing LC80 TCAGGCGGGATGAAGATGAT 56 PCR verification LC153GCTGGGATACGCTGGTGTTTA 57 PCR verification LC154 CACAGCGCAAGGACGTTGA 58PCR verification LC155 ACACAACATGACGGGCTT 59 PCR verificationThe SEQ ID Nos: 29 to 59 in table 3 correspond to SEQ ID Nos: 52 to 82of the priority application.

The ability of Cas9 to kill bacteria when directed to cut in theirchromosome has been used as a counter-selection tool for the purpose ofgene editing and for the development of sequence-specific antimicrobials[7, 14, 15]. However, the mechanism of Cas9-mediated cell death has sofar remained unclear. Here the inventors shown that not all targets areequal and E. coli can survive active targeting at some positions.Cas9-induced breaks activate the SOS response and can be repaired by theHDR pathway. This enables E. coli to tolerate the presence of weakself-targeting CRISPR systems. Other targets can be cleaved efficientlyleading to the introduction of DSB in all copies of the chromosomesimultaneously. In the absence of a template for HDR, extensiverecession of the DNA ends by RecBCD and other nucleases is likely thecause of cell death.

Variations in the efficiency of Cas9 cleavage between different targetshave been reported previously [10,26,27]. The ability to predict theefficacy of guide RNAs is of prime importance for all applications ofCas9 technologies. High-throughput screens of sgRNA libraries in humanor mouse cells have allowed identifying good targets[26,28], and wereused to build predictive models for the design of highly active sgRNAs.However, the most recent model from Jong and colleagues [28] gave verypoor prediction for the activity of the 13 targets that were used in ourstudy. This could stem from differences in the requirements forefficient Cas9 targeting between mammalian cells and E. coli, as well asthe fact that these screens were performed using sgRNAs instead of thedual crRNA and tracrRNA system. In particular some features thatinfluence the expression of the sgRNA, loading of the sgRNA on Cas9, orthe accessibility of the target DNA are likely not generalizable topresent system. This highlights the necessity to perform similar screensin bacteria. The inventors demonstrate here that the level of SOSinduction can be used to estimate the efficiency of Cas9 interference inE. coli, with good targets showing a more pronounced SOS response (FIG.3B). This might be useful to score candidate targets and could also beused in combination with Fluorescence-Activated Cell Sorting (FACS) toscreen for highly active guides in a library. A better knowledge of whatmakes a good CRISPR target will be critical for the development ofreliable genome engineering tools as well as CRISPR antimicrobials.

Interestingly cell death is not the only possible outcome of efficientCas9 cleavage in the chromosome of E. coli. Large deletions can beintroduced through recombination between distant homologous sequences.This is consistent with rearrangements observed in a previous studywhere a mRFP gene integrated in the genome was targeted by Cas9 [20].

The pCas9 plasmid carrying either an empty CRISPR array, the lacZ1spacer or the lacZ2 spacer was transformed in cells containing the pLC13plasmid which carries the Mu gam gene under the control of a pBADpromoter. Transformants were plated on selective medium either with orwithout arabinose (−ara/+ara). The results are shown in FIG. 6A-B. UponMu-Gam induction with arabinose, Cas9 killing efficiency using the weaklacZ1 spacer is increased more than 1000×. A more moderated increase inkilling efficiency is also observed when targeting with the strongerlacZ2 spacer.

Example 10

The inventors have developed an inducible Cas9-sgRNA system targetingthe E. coli chromosome with very low leakiness and high cleavingefficiency. This setup allows for a 3-log difference in cell survival inthe presence of inducer with virtually no difference in the amount ofviable cells in its absence. In this architecture, Cas9 expression isunder the control of the PhIF repressor (1), which can be activated uponaddition of a small molecule, 2,4-diacetylphloroglucinol (DAPG). Thetranscription of the sgRNA, which targets a genomic sequence at the 5′end of the lacZ gene, is under the control of a synthetic constitutivepromoter, PJ23108. Both elements are encoded in a low copythermosensitive origin of replication, pSC101* (FIG. 7).

The inventors show that the co-expression of Mu-Gam, a viral proteinthat inhibits the host's homologous recombination machinery, can serveas an adjuvant to increase Cas9-mediated killing when targeting thebacterial chromosome. The effects can increase the efficiency ofCas9-mediated cell death by 15-200 fold. These results have beendemonstrated for different crRNA sequences, especially when they are notoptimized. This system implements a different architecture, relying onthe tightly regulated expression of Cas9 as well as a constitutivelytranscribed sgRNA that targets a genomic sequence. The inventorsassessed if the addition of Mu- and Lambda-Gam proteins to this systemimproves the efficiency of Cas9-mediated killing of target bacteria.

This approach is important, since there exist a variety of conditionswhere cleavage may be suboptimal as compared to in vitro assays. Eventhough laboratory experiments show that invention's current Cas9-sgRNAdesign allows for a 3-log killing upon induction, the conditions mayvary in other setups; for instance, natural SNPs of the target sequenceor escape mechanisms due to mutations in the targeted sequence canreduce the efficiency of Cas9 cleavage; non-optimally designed sgRNAs ortargeting a heterogeneous population; protein expression inducers maynot be efficiently administered or show toxicity in different setups,such as in vivo models, reducing expression levels of Cas9 and henceefficiency; and finally, the physiological state of the cell mayinfluence the expression levels and cleavage efficiency of Cas9: in alaboratory setup, cells are typically maintained in the log growthphase, while in many other situations they may enter different growthregimes (such as stationary phase). For all these situations, anadjuvant for Cas9 activity will be beneficial to achieve the desiredeffects.

A) Use Non-Optimally Designed sgRNA Sequences to Reduce Cas9 EfficiencyEven in the Presence of Maximal Amounts of Inducer.

It has been shown that the Cas9-sgRNA machinery can tolerate mismatchesat the 5′ end of the sgRNA in the targeted genomic sequence, althoughwith reduced cleavage efficiency. To do this, the inventors constructedvariants of the plasmid pPhIF-Cas9 possessing sequential mutations inthe first 5 nucleotides at the 5′ end of the sgRNA. The cleavageefficiency of these variants was assessed in LB-agar plates by thedroplet method at different concentrations of DAPG. These plasmidvariants were used in subsequent experiments to assess the effect of theMu-Gam and the Lambda-Gam proteins in suboptimal cleavage conditionscaused by non-optimized sgRNA sequences.

B) Optimize Mu- and Lambda-Gam Expression Levels.

The inventors verified that a defined expression level exists for theMu/Lambda-Gam proteins to act as adjuvants of Cas9-mediated killingwhile proving non-toxic upon expression on their own. In an initial stepto facilitate the characterization and further engineering of thesystem, several RBS sequences for the Mu-Gam protein were screened in aseparate plasmid, pBAD-MuGam (SEQ ID NO: 69):

The RBS sequence upstream of the mu-gam gene in pBAD-MuGam (FIG. 8) wasmodified by running an iPCR reaction on the plasmid followed by aone-pot phosphorylation-ligation reaction. The religated plasmids wereco-transformed into MG1655 cells containing the pPhIF-Cas9 plasmid,plated in LB-agar supplemented with 50 μg/mL kanamycin, 100 μg/mLchloramphenicol, 0.1 mM IPTG and 40 μg/mL X-gal and grown for 20 hoursat 30° C. Next, 95 single colonies were selected and grown in 500 μL LBsupplemented with 50 μg/mL kanamycin and 100 μg/mL chloramphenicol in96-deep-well plates for 18 hours at 1000 rpm at 30° C. Next day, eachculture was diluted 1:100 in distilled water and assayed by the dropletmethod in LB agar plates. Briefly, individual 8 μL droplets were platedonto the surface of LB-agar plates supplemented with 50 μg/mL kanamycin,25 μg/mL chloramphenicol, 0.1 mM IPTG and 40 μg/mL X-gal. The plateswere then gently turned in a vertical position to allow the droplets toslide down the surface of LB-agar and incubated o/n at 30° C. for 18hours. The cells were assayed in four conditions: plates withoutinducer; plates that contained 5 mM arabinose; plates that contained 0.1mM DAPG; and plates that contained both 5 mM arabinose and 0.1 mM DAPG.This experiment allows for the comparison of cell morphology and/ortoxicity in the presence of Mu-Gam only and its effects when Cas9-sgRNAis co-expressed (FIG. 9).

The initial RBS screening yielded several clones that had altered cellmorphology and appearance (smaller and translucent) in the presence ofboth Mu-Gam and Cas9-sgRNA while showing a normal aspect in the presenceof Mu-Gam only. These clones were also verified for Cas9-sgRNA activityand achieved similar killing efficiencies as the pPhIF-Cas9 systemalone.

The inventors selected one clone based on its potential Mu-Gam adjuvantactivity and performed a more detailed characterization on LB-agarplates in the same four conditions described above (no inducer; plusarabinose; plus DAPG; plus DAPG and arabinose). After a 24-hourincubation period, massive cell death occurred, which was especiallypronounced in cells that were plated at a higher density, as can be seenin FIG. 10. For the “+DAPG, +Ara” dataset, colonies were directlycounted from the undiluted droplet. For the “+DAPG” dataset, colonieswere counted at 10⁻² and 10⁻³ dilutions, the dilution factor calculatedand the number of CFUs in the undiluted droplet estimated. For “Noinducer” and “+Ara” conditions, the number of CFUs in the uniduluteddroplet was estimated by counting the number of colonies in the 10⁻⁵ and10⁻⁶ dilutions and the dilution factor calculated. Moreover, if the sameexperiment is performed in cells containing pBAD-MuGam and a pPhIF-Cas9variant with a sgRNA not targeting the genome, no cell death is seen forany conditions (FIG. 11). Cells containing pBAD-MuGam hit wereco-transformed with a pPhIF-Cas9 variant with a non-targeted sgRNAsequence. Cells were analyzed by the droplet method as explained in (A)and CFUs counted. To estimate the CFUs in the undiluted droplet, CFUswere counted at the 10⁻⁶ and 10⁻⁵ dilutions, the dilution factorcalculated and the number of CFUs in the undiluted droplet calculated.These results indicate that expression of Gam together with a targetedCas9-sgRNA system leads to improved cell killing, in an assay whereCas9-mediate killing is already very efficient in itself. This assay wasalso performed under sub-optimal targeting conditions through theintroduction of mismatches between the guide RNA and the target.Additionally, the same experiments can be performed with Lambda-Gam byconstructing the plasmid pBAD-LambdaGam (FIG. 12):

C) Construction of an Integrated Architecture Encoding Cas9-sgRNA andMu/Lambda-Gam.

Both Cas9-sgRNA and Mu/Lambda-Gam inducible cassettes can be integratedin the same plasmid containing a low copy origin of replication (pSC101)as well as a cos site. This architecture possesses two advantages: a lowcopy origin of replication allows for a wider tunable range of RBSstrengths and reduced leakiness, hence increasing the expression spacefor a given protein; and also offers a platform for generating packagedcosmids to transduce the genetic program into a target strain. Theintegrated vectors, pCas9-MuGam and pCas9-LambdaGam, are shown on FIG.13.

The expression levels of the Mu/Lambda-Gam proteins was tuned andcharacterized as described in (B) in MG1655 using transformed cells as atestbed.

D) Packaging of pCas9-MuGam and pCas9-LambdaGam into Cosmid Particles.

Once optimal expression levels for Mu-Gam and Lambda-Gam have been foundas described in (C), the inventors performed transduction experimentswith the packaged cosmid particles. To do this, the optimizedpCas-MuGam/Lambda-Gam plasmids was transformed in CY2120 cells, platedon LB-agar plus 50 μg/mL kanamycin and incubated o/n at 30° C. A singlecolony was picked and grown in liquid LB to an OD600 of 0.5 at 30° C. Toinduce the packaging, the culture was heat-shocked at 42° C. for 20minutes and subsequently incubated at 37° C. for 4 hours. Cells wereharvested, resuspended in lambda dilution buffer and lysed by addingchloroform. The packaged cosmid was isolated from the supernatant bycentrifugation to pellet cell debris. The titer of the packaged cosmidwas then determined by transduction of E. coli DH5-alpha.

Both pPhIF-Cas9 and pCas9-MuGam or pCas9-LambdaGam cosmids weregenerated and assayed in parallel to assess the efficiency ofCas9-mediated cell death and the effects of the addition of one of theviral proteins.

E) Pathogenic E. coli Strains.

Finally, the same tests are performed in pathogenic E. coli strains ThesgRNA variant used in all experiments described above also targets thegenome of E. coli LF82, a known human pathogen. The efficiency of theengineered cosmids was assessed in this bacterial strain and can bepotentially expanded to many other known human pathogens.

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1-27. (canceled)
 28. A method for killing a bacterium comprisingcontacting the bacterium with at least one recombinant phagemid(s) orplasmid(s); wherein the recombinant phagemid(s) or plasmid(s) encodes anendonuclease that creates a double-stranded break (DSB) in thechromosomal or extrachromosomal DNA of the bacterium, and an exogenousprotein that inhibits DSB repair.
 29. The method of claim 28, whereinthe exogenous protein is encoded by the same vector as the endonucleaseor by a separate vector.
 30. The method of claim 28, wherein the proteinis synthetized before contacting with the bacterium.
 31. The method ofclaim 28, wherein the endonuclease is selected from a meganuclease or anartificial endonuclease.
 32. The method of claim 28, wherein theendonuclease specifically cleaves the chromosomal or extrachromosomalDNA of the bacterium at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10different sites.
 33. The method of claim 28, wherein the at least onerecombinant phagemid(s) or plasmid(s) encodes a Cas9 nuclease, a guideRNA, and an exogenous protein that inhibits DNA repair selected from thegroup consisting of a Mu phage Gam protein, a lambda phage Gam protein,and a phage T7 gp5.9 protein.
 34. The method of claim 28, wherein the atleast one recombinant phagemid(s) is selected from the group consistingof M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1Puna-like, P2, 13, Bcep1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3 phage, P37,MS2, Qβ, or Phi X 174, T2 phage, T12 phage, R17 phage, M13 phage, G4phage, Enterobacteria phage P2, P4 phage, N4 phage, Pseudomonas phageϕ6, ϕ29 phage and 186 phage.
 35. The method of claim 28, wherein thebacterium comprises a recBCD homologous repair pathway or addAB system.36. The method of claim 28, wherein the bacterium is selected from thegroup consisting of Enterobacter, Streptococci, Staphylococci,Enterococci, Salmonella, Pseudomonas, and Mycobacterium.
 37. The methodof claim 28 wherein the recombinant phagemid(s) or plasmid(s) encode(s)an endonuclease that creates a double-stranded break (DSB) in anantibiotic resistance gene encoded by the bacterium
 38. λ phagemid orplasmid vector encoding an endonuclease and an exogenous proteininhibiting DSB repair.
 39. The phagemid or plasmid vector of claim 38wherein the recombinant phagemid(s) is selected from the groupconsisting of M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1Puna-like, P2,13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3phage, P37, MS2, Qβ, or Phi X 174, T2 phage, T12 phage, R17 phage, M13phage, G4 phage, Enterobacteria phage P2, P4 phage, N4 phage,Pseudomonas phage ϕ6, ϕ29 phage and 186 phage.
 40. The phagemid orplasmid vector of claim 38, wherein the phagemid vector is a P1bacteriophage.
 41. The phagemid or plasmid vector of claim 38, whereinthe phagemid vector is a λ bacteriophage.
 42. A pharmaceuticalcomposition comprising a phagemid or plasmid vector encoding anendonuclease, and an exogenous protein inhibiting DSB repair or a vectorencoding an exogenous protein inhibiting DSB repair, and apharmaceutically acceptable vehicle.
 43. The pharmaceutical compositionof claim 42 further comprising an antibiotic.
 44. The pharmaceuticalcomposition of claim 42 containing a phagemid or plasmid vector encodingan endonuclease and a vector encoding an exogenous protein inhibitingDSB repair.
 45. The pharmaceutical composition of claim 42, wherein saidexogenous protein is encoded by the same vector as the endonuclease.